Current mass spectrometric research into biopolymers such as peptides, proteins and genetic material is frequently coupled with fast separation methods such as liquid chromatography (HPLC or simply LC) or capillary electrophoresis (CE). The objective here is often to fragment biopolymer ions in the mass spectrometer in order to obtain information about the sequences of the biopolymer building blocks; for peptides and proteins this means information about the sequence of the amino acids. Therefore, fragment ion spectra have to be acquired. The mass spectrometers required for this are known as “tandem mass spectrometers”. The methods of acquiring fragment ion spectra with tandem mass spectrometers are often abbreviated to MS/MS.
Tandem mass spectrometers comprise an initial mass spectrometer to select ions of a certain type, a fragmentation device, in which these ions are fragmented, and another mass spectrometer to analyze the fragment ions. In ion trap mass spectrometers, these processes for selecting, fragmenting and analyzing the fragment ions can be performed time-sequentially in the same ion trap. This is then termed “tandem-in-time”, in contrast to “tandem-in-space” in the case of spatially separated mass spectrometers.
In proteomics it is frequently necessary to analyze thousands of peptides which have been obtained from an enzymatic digest of a complex protein mixture. If in the following the objective of the invention is described, it is particularly with respect to these very complex peptide mixtures.
The upstream separation for the biopolymers provides a certain analyte substance, in this specific case a digest peptide, for only a few seconds to the mass spectrometer. For such cases, several commercial companies supply tandem mass spectrometers equipped with measurement procedures for the automatic acquisition of fragment mass spectra. Mass spectra are acquired in continuous sequence, between one and twenty mass spectra per second in fast mass spectrometers, for example. For each mass spectrum, an evaluation program is then used to determine in real time whether, if at all, one or several digest peptides are provided in sufficient concentration. With the complex mixtures described above, several digest peptides are often supplied at the same time; frequently even as many as ten to twenty digest peptides simultaneously.
In this case, a real time mathematical analysis of the mass spectrum is carried out first to select which ionic species is to be fragmented for the acquisition of a fragment ion spectrum. Doubly charged ions are best suited to fragmentation, and therefore the most intensive ionic species which occurs with a double charge within a predetermined mass range, not occurring in an exclusion table, is generally used. The exclusion table contains the mass values of those peptides which have already been analyzed in previous measuring cycles or which have been marked as not of interest from the outset. This is followed by a further spectrum acquisition in which the ionic species selected is isolated by separation in the first mass spectrometer and fragmented in the fragmentation stage; the fragment ions are then measured as a fragment ion spectrum. There are various methods of fragmentation whose parameters are generally set blind to the settings that have, on average, proven favorable for ions of a digest peptide of the mass in question.
The various parameters for the fragmentation method differ widely for different mass spectrometers and different types of fragmentation. A widely used fragmentation method is collisionally induced fragmentation (CID), in which collisions with a collision gas transfer energy to the ion. Depending on the collision energy, this may lead to a fragmentation after just one collision, or it may require a large number of collisions resulting in different types of fragment ion spectra. Another type of fragmentation, offered in some mass spectrometers, is the fragmentation by low-energy electrons, either by direct bombardment or by transfer of the electrons from negatively charged ions or highly excited neutral particles (ECD=electron capture dissociation; ETD=electron transfer dissociation, MAID=metastable atom-induced dissociation). For these types of fragmentation by electrons, the only parameters are those of the electron density used and the irradiation time.
In mass spectrometers equipped with quadrupole collision cells, the fragmentation parameters are, in particular, the collision energy with which the selected ions are injected into the gas-filled collision cell and, in some mass spectrometers, also the type of collision gas, which cannot be changed quickly, and certainly not from spectrum acquisition to spectrum acquisition. These instruments with collision cell include the triple quadrupole mass spectrometer (generic abbreviation QqQ), and also certain types of time-of-flight mass spectrometer with orthogonal ion injection (generic abbreviation QqOTOF). With time-of-flight mass spectrometers with orthogonal ion injection it is also possible to change the duration of the spectrum acquisition, because they acquire individual spectra with high spectrum acquisition rate and continuously add the spectra together to form a single sum spectrum.
For mass spectrometers comprising RF quadrupole ion traps, in which a collisionally induced fragmentation is carried out using helium as the damping and fragmentation gas, there are essentially only two parameters: filling time and fragmentation time. The excitation RF voltage level here is always chosen so that the selected ions excited to oscillations just avoid hitting the end cap electrodes. This excitation RF voltage is therefore not generally available as a variable parameter.
Unfortunately it very often appears when using these fragment ion spectra in identity and structure searches by “search engines” in protein sequence databases, the quality of the spectra is not sufficiently high. Analyses show that, in some types of mass spectrometer, the proportion of fragment ion spectra with adequate quality is frequently no more than ten percent. In a three-hour run of a single liquid chromatographic separation with mass spectrometric analysis, some 20,000 to 60,000 fragment ion spectra can be acquired, and so the absolute number of 2,000 to 6,000 qualitatively good spectra often seems to be pleasingly high; however, the analytical objective of recording as many analyte substances as possible is by far not adequately achieved. The same occurs with capillary electrophoretic separation.
Under the best conditions available in tandem mass spectrometry, 20 to 30 percent of the spectra obtained have sufficient quality, still a very low and unsatisfying proportion.